目的:剥脱综合征(XFS)是一种富含弹性蛋白组织的全身性疾病,涉及眼前房中纤维状剥脱物质(XFM)的沉积,可以促进青光眼。这项研究的目的是创建具有CRISPR/Cas9诱导的从人类全基因组关联研究(GWAS)鉴定的候选基因变异的小鼠,并筛选它们的XFS指数。
方法:在Agpat1、Cacna1a、Loxl1,Pomp,Rbms3,Sema6a,和使用基于CRISPR/Cas9的基因编辑的C57BL/6J小鼠的Tlcd5基因。菌株通过裂隙灯进行表型分析,SD-OCT成像,和1-5岁的眼底检查。还研究了12mos大小鼠的较小队列。
结果:在六个靶标中鉴定出有害的变体;Pomp难以靶向。分离了一些靶标的多个等位基因,产生12株。在所有基因型和年龄中,通过902次裂隙灯检查评估277只小鼠,928次SD-OCT检查,还有358次眼底检查.Agpat1或Cacna1a突变的纯合性导致早期致死;Loxl1突变的纯合性导致盆腔器官脱垂,防止衰老。Loxl1纯合子表现出与XFS潜在相关的结膜表型。多种其他基因型特异性表型被不同地鉴定。在任何小鼠中均未观察到XFM。
结论:本研究在任何菌株中均未检测到XFM。这可能是由于物种特异性差异,背景依赖性,或老化不足。或者,有可能目前的候选人,根据与GWAS信号的接近度选择,不是通过单基因功能丧失机制起作用的效应子。
OBJECTIVE: Exfoliation syndrome (XFS) is a systemic disease of elastin-rich tissues involving a deposition of fibrillar exfoliative material (XFM) in the anterior chamber of the eye, which can promote glaucoma. The purpose of this study was to create mice with CRISPR/Cas9-induced variations in candidate genes identified from human genome-wide association studies (GWAS) and screen them for indices of XFS.
METHODS: Variants predicted to be deleterious were sought in the Agpat1, Cacna1a, Loxl1, Pomp, Rbms3, Sema6a, and Tlcd5 genes of C57BL/6J mice using CRISPR/Cas9-based gene editing. Strains were phenotyped by slit-lamp, SD-OCT imaging, and fundus exams at 1-5 mos of age. Smaller cohorts of 12-mos-old mice were also studied.
RESULTS: Deleterious variants were identified in six targets; Pomp was recalcitrant to targeting. Multiple alleles of some targets were isolated, yielding 12 strains. Across all genotypes and ages, 277 mice were assessed by 902 slit-lamp exams, 928 SD-OCT exams, and 358 fundus exams. Homozygosity for Agpat1 or Cacna1a mutations led to early lethality; homozygosity for Loxl1 mutations led to pelvic organ prolapse, preventing aging. Loxl1 homozygotes exhibited a conjunctival phenotype of potential relevance to XFS. Multiple other genotype-specific phenotypes were variously identified. XFM was not observed in any mice.
CONCLUSIONS: This study did not detect XFM in any of the strains. This may have been due to species-specific differences, background dependence, or insufficient aging. Alternatively, it is possible that the current candidates, selected based on proximity to GWAS signals, are not effectors acting via monogenic loss-of-function mechanisms.